Analysis of unsaturated C₂₇ sterols by nuclear magnetic resonance spectroscopy

Complete ¹H and ¹³C nuclear magnetic resonance (NMR) signal assignments have been established for 5α-cholestan-3β-ol, 22 unsaturated C₂₇ sterols, and their acetate derivatives. Assignments were made from a combination of 1D and 2D spectra and include stereochemical ¹H assignments for the C-22 and C-23 protons of 5α-cholesta-8,24-dien-3β-ol and other Δ²⁴ sterols with a C₈ side chain. At the temperature and concentration range described, chemical shifts were generally reproducible to ±0.01 ppm in ¹³C spectra and ±0.001 ppm in ¹H spectra. Except for some overlapped or strongly coupled ¹H resonances, chemical shifts are given to these precisions, which are an order of magnitude better than for most data given previously. Full ¹H NMR data have been reported previously for only three of the 46 compounds, and ¹³C data were unavailable for many, including the previously undescribed cholesta-5,8(14)-dien-3β-ol. An extensive set of ¹H- ¹H coupling constants for the unsaturated sterols indicated considerable conformational diversity, which was confirmed by molecular modeling. The conformational diversity together with other factors led to a complex pattern of ¹³C substituent-induced chemical shifts (SCS) that appeared to elude any simple empirical correlations with structure. By contrast, the ¹H SCS correlated reasonably well with simple structural features. The high precision of the SCS revealed small but measurable effects of a double bond on ¹H resonances up to 12 bonds away. Also discussed are the utility and limitations of NMR for the identification of unsaturated sterols, estimation of purity, and analysis of mixtures, with an emphasis on special problems encountered at a microgram level.